Hepatocyte nuclear factor 4α mediated quinolinate phosphoribosylltransferase (QPRT) expression in the kidney facilitates resilience against acute kidney injury.

Nicotinamide adenine dinucleotide (NAD+) levels decline in experimental models of acute kidney injury (AKI). Attenuated enzymatic conversion of tryptophan to NAD+ in tubular epithelium may contribute to adverse cellular and physiological outcomes. Mechanisms underlying defense of tryptophan-dependent NAD+ production are incompletely understood. Here we show that regulation of a bottleneck enzyme in this pathway, quinolinate phosphoribosyltransferase (QPRT) may contribute to kidney resilience. Expression of QPRT declined in two unrelated models of AKI. Haploinsufficient mice developed worse outcomes compared to littermate controls whereas novel, conditional gain-of-function mice were protected from injury. Applying these findings, we then identified hepatocyte nuclear factor 4 alpha (HNF4α) as a candidate transcription factor regulating QPRT expression downstream of the mitochondrial biogenesis regulator and NAD+ biosynthesis inducer PPARgamma coactivator-1-alpha (PGC1α). This was verified by chromatin immunoprecipitation. A PGC1α - HNF4α -QPRT axis controlled NAD+ levels across cellular compartments and modulated cellular ATP. These results propose that tryptophan-dependent NAD+ biosynthesis via QPRT and induced by HNF4α may be a critical determinant of kidney resilience to noxious stressors.

Lactate-induced activation of HCA2 improves survival in mice with sepsis.

Sepsis is characterized by systemic inflammation that is caused by infection and by activation of proinflammatory pathways, resulting in mitochondrial and cellular dysfunction leading to multiorgan failure. Here, we show the following: 1) in peritoneal immune cells, particularly macrophages, from mice that have undergone cecal ligation and puncture (CLP), hydroxycarboxylic acid receptor 2 (HCA2) expression increased in parallel with proinflammatory cytokines; 2) post-CLP survival rates of Hca2-/- knockout mice (n = 22) were lower than those of wild-type (WT) mice (n = 15) (P < 0.011), which is suggestive of a protective role for HCA2 in sepsis; 3) WT mice subjected to CLP-induced sepsis and treated with lactated Ringer's solution (LR, n = 13) survived longer than those treated with normal saline (n = 14; P < 0.027); 4) LR treatment of CLP-induced sepsis reduced proinflammatory cytokine expression in CD11b+F4/80+ macrophages and promoted M2-like polarization; 5) HCA2 was expressed by kidney in the setting of sepsis, but not by normal kidneys; 6) LR administration attenuated sepsis-associated acute kidney injury (AKI), partly restored expression of the key regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1α (P < 0.03), and reduced proinflammatory cytokine production (TNF-α, P < 0.04; IL-1ß, P < 0.006; IL-6, P < 0.03). Our data suggest that lactate-induced activation of HCA2 during sepsis activates a negative feedback loop to attenuate the inflammatory response. The data further suggest that fluid resuscitation with LR may benefit patients with sepsis, particularly those with sepsis-associated AKI treated with potentially lactate-depleting renal replacement therapies.-Takakura, A., Zandi-Nejad, K. Lactate-induced activation of HCA2 improves survival in mice with sepsis.

Insulin and SGK1 reduce the function of Na+/monocarboxylate transporter 1 (SMCT1/SLC5A8).

SMCTs move several important fuel molecules that are involved in lipid, carbohydrate, and amino acid metabolism, but their regulation has been poorly studied. Insulin controls the translocation of several solutes that are involved in energetic cellular metabolism, including glucose. We studied the effect of insulin on the function of human SMCT1 expressed in Xenopus oocytes. The addition of insulin reduced α-keto-isocaproate (KIC)-dependent 22Na+ uptake by 29%. Consistent with this result, the coinjection of SMCT1 with SGK1 cRNA decreased the KIC-dependent 22Na+ uptake by 34%. The reduction of SMCT1 activity by SGK1 depends on its kinase activity, and it was observed that the coinjection of SMCT1 with S442D-SGK1 (a constitutively active mutant) decreased the KIC-dependent 22Na+ uptake by 50%. In contrast, an SMCT1 coinjection with K127M-SGK1 (an inactive mutant) had no effect on the KIC-dependent Na+ uptake. The decreasing SMCT1 function by insulin or SGK1 was corroborated by measuring [1-14C]acetate uptake and the electric currents of SMCT1-injected oocytes. Previously, we found that SMCT2/Slc5a12-mRNA, but not SMCT1/Slc5a8-mRNA, is present in zebrafish pancreas (by in situ hybridization); however, SLC5a8 gene silencing was associated with the development of human pancreatic cancer. We confirmed that the mRNA and protein of both transporters were present in rat pancreas using RT-PCR with specific primers, Western blot analysis, and immunohistochemistry. Additionally, significant propionate-dependent 22Na+ uptake occurred in pancreatic islets and was reduced by insulin treatment. Our data indicate that human SMCT1 is regulated by insulin and SGK1 and that both SMCTs are present in the mammalian pancreas.

The role of HCA2 (GPR109A) in regulating macrophage function.

We investigated the novel role of HCA2 (GPR109A) and its ligand nicotinic acid in regulating macrophage function. Hca2 expression in the RAW264.7 murine macrophage cell line is strongly induced by LPS treatment and correlates with the expression of TNF-α. Treatment with 300 µM nicotinic acid (reported EC50 3 µM, peak plasma concentration 50-300 µM), significantly inhibited TNF-α, IL-6, IL-12p40, and IL-1ß production (P<0.05) in LPS (1 ng/ml)-stimulated wild-type murine bone marrow-derived macrophages (BMMs) but failed to do so in Hca2(-/-) BMMs. Treatment with nicotinic acid reduced nuclear factor κB (NF-κB) activation levels by 43% (P<0.03) in wild-type BMMs 6 h after LPS stimulation but not in Hca2(-/-) BMMs. Nicotinic acid significantly inhibited wild-type BMM chemotaxis (P<0.001), but had no effect on the chemotaxis of Hca2(-/-) BMMs. A significant increase in low-density lipoprotein uptake by both wild-type (P<0.006) and Hca2(-/-) BMMs (P<0.03) in response to LPS was observed, which was significantly suppressed by nicotinic acid in wild-type BMMs (P<0.04) but not in Hca2(-/-) BMMs. Our results suggest that the nicotinic acid-HCA2 axis is a novel negative regulator of macrophage activation.

Pyrimethamine inhibits adult polycystic kidney disease by modulating STAT signaling pathways.

Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder mostly caused by mutations in PKD1, encoding polycystin-1 (PC1). The disease is characterized by development and growth of epithelium-lined cyst in both kidneys, often leading to renal failure. There is no specific treatment for this disease. Here, we report a sustained activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in ischemic injured and uninjured Pkd1 knockout polycystic kidneys and in human ADPKD kidneys. Through a chemical library screen, we identified the anti-parasitic compound pyrimethamine as an inhibitor of STAT3 function. Treatment with pyrimethamine decreases cell proliferation in human ADPKD cells and blocks renal cyst formation in an adult and a neonatal PKD mouse model. Moreover, we demonstrated that a specific STAT3 inhibitor, S3I-201, reduces cyst formation and growth in a neonatal PKD mouse model. Our results suggest that PC1 acts as a negative regulator of STAT3 and that blocking STAT3 signaling with pyrimethamine or similar drugs may be an attractive therapy for human ADPKD.

Ischemic injury enhances dendritic cell immunogenicity via TLR4 and NF-kappa B activation.

Ischemic (isc) injury during the course of transplantation enhances the immunogenicity of allografts and thus results in poorer graft outcome. Given the central role of dendritic cells (DCs) in mounting alloimmune responses, activation of donor DCs by ischemia may have a primary function in the increased immunogenicity of isc allografts. In this study, we sought to investigate the effect of ischemia on DC activity in vitro. Following induction of ischemia, bone marrow-derived DCs were shown to augment allogeneic T cell proliferation as well as the IFN-gamma response. Isc DCs produced greater levels of IL-6, and isc insult was concurrent with NF-kappaB activation. TLR4 ligation was also shown to occur in isc DCs, most likely in response to the endogenous ligand heat shock protein 70, which was found to be elevated in DCs following isc injury, and lack of TLR4 abrogated the observed effects of isc DCs. As compared with control DCs, isc DCs injected into the footpads of mice demonstrated enhanced migration, which was concomitant with increased recipient T cell activity. Moreover, isc DCs underwent a greater degree of apoptosis in the lymph nodes of injected mice, which may further demonstrate enhanced immunogenicity of isc DCs. We thus show that isc injury of DCs enhances DC function, augments the allogeneic T cell response, and occurs via ligation of TLR4, followed by activation of NF-kappaB. These data may serve to identify novel therapeutic targets to attenuate graft immunogenicity following ischemia.

Slc26a9 is inhibited by the R-region of the cystic fibrosis transmembrane conductance regulator via the STAS domain.

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl(-)-HCO(3)(-) exchanger and Cl(-) channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl(-)-HCO(3)(-) exchange and Cl(-) currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-DeltaSTAS) virtually eliminated the Cl(-) currents with only a modest affect on nCl(-)-HCO(3)(-) exchange activity. Co-expression of a9-DeltaSTAS and the (R)CFTR fragment did not alter the residual a9-DeltaSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.

Slc26a9--anion exchanger, channel and Na+ transporter.

The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl(-)-HCO(3)(-) exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO(3)(-) secretion; Na(+) electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl(-)-HCO(3)(-) exchanger, Cl(-) channel, and Na(+)-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues.

Mechanisms of gut microbiota-mediated bone remodeling.

The mechanisms underlying the systemic effects mediated by gut microbiota are under active investigation. In addition to local, direct effects of gut microbiota on the host, metabolic products from microbiota may act peripherally, reaching distal organs through the circulation. In our recent publication we demonstrated that gut microbiota influence bone remodeling distally, promoting both bone resorption and formation. We proposed that these effects are mediated, at least in part, by the induction of insulin like growth factor (IGF-1) by the microbiota metabolite short chain fatty acids (SCFA). Here we explore additional mechanisms by which microbial metabolites could directly or indirectly alter host bone remodeling. We discuss whether SCFA directly modulate bone resorption by their actions on osteoclasts, and test the possibility that serotonin is another gut microbiota derived long-distance mediator of effects on bone remodeling. A detailed understanding of the mechanisms of microbiota effect on bone remodeling could help establish potential therapeutic strategies to promote bone health.

De novo thrombotic microangiopathy after kidney transplantation.

Thrombotic microangiopathy (TMA) is a serious complication of transplantation that adversely affects kidney transplant recipient and allograft survival. Post-transplant TMA is usually classified into two categories: 1) recurrent TMA and 2) de novo TMA. Atypical hemolytic uremic syndrome (aHUS) resulting from dysregulation and over-activation of the alternate complement pathway is a rare disease but the most common diagnosis associated with recurrence in the allografts. De novo TMA, on the other hand, represents an overwhelming majority of the cases of post-transplant TMA and is a substantially more heterogeneous entity than recurrent aHUS. Here, we review the etio-pathogenesis, diagnosis and treatment options for de novo post-transplant TMA. It is usually in the setting of calcineurin inhibitor use, mammalian target of rapamycin inhibitor use, or antibody mediated rejection; recently genetic mutations in complement regulatory genes for Factor H and Factor I similar to those described in aHUS have been reported in up to a third of these patients. Systemic signs of TMA are frequently absent, and a renal allograft biopsy is often needed to establish the diagnosis. Although withdrawal of the offending agents is usually the first line of treatment and resolution of laboratory abnormalities has been documented with this approach in several case reports and case series, available retrospective data demonstrate lack of benefit in long-term graft outcomes. Co-stimulation blockage with belatacept provides an effective alternate immunosuppressive strategy for these patients. Anti-complement therapy with eculizumab is effective in some cases; further work is required to define which patients with TMA (with and without concomitant antibody-mediated rejection) would benefit from receiving this treatment, and what biomarkers can be used to identify them.

Uricosuric targets of tranilast.

Uric acid, generated from the metabolism of purines, has both proven and emerging roles in human disease. Serum uric acid in humans is determined by production and by the net balance of reabsorption and secretion in kidney and intestine. In the human kidney, epithelial reabsorption dominates over secretion, such that in normal subjects there is at least 90% net reabsorption of filtered urate resulting in a fractional excretion of <10%. Tranilast, an anti-inflammatory drug with pleiotropic effects, has a marked hypouricemic, uricosuric effect in humans. We report here that tranilast is a potent inhibitor of [14C]-urate transport mediated by the major reabsorptive urate transporters (URAT1, GLUT9, OAT4, and OAT10) in Xenopus oocytes; this provides an unequivocal molecular mechanism for the drug's uricosuric effect. Tranilast was found to inhibit urate transport mediated by URAT1 and GLUT9 in a fully reversible and noncompetitive (mixed) manner. In addition, tranilast inhibits the secretory urate transporters NPT1, OAT1, and OAT3 without affecting the secretory efflux pump ABCG2. Notably, while benzbromarone and probenecid inhibited urate as well as nicotinate transport, tranilast inhibited the urate transport function of URAT1, GLUT9, OAT4, OAT10, and NPT1, without significantly affecting nicotinate transport mediated by SMCT1 (IC 50 ~1.1 mmol/L), SMCT2 (IC 50 ~1.0 mmol/L), and URAT1 (IC 50 ~178 µmol/L). In summary, tranilast causes uricosuria by inhibiting all the major reabsorptive urate transporters, selectively affecting urate over nicotinate transport. These data have implications for the treatment of hyperuricemia and gout, the pharmacology of tranilast, and the structure-function analysis of urate transport.

Physiology of electrogenic SLC26 paralogues.

SLC26 anion exchangers transport monovalent and divalent anions, with a diversity of anion specificity and stoichiometry. Our microelectrode studies indicate that several SLC26 members are electrogenic. We reported that Slc26a6 functions as a Cl-/formate, Cl-/oxalate, Cl-/OH- and electrogenic Cl-/nHCO3- exchanger. Recently, we have also confirmed that Slc26a7 does not behave as a Cl-/HCO3- exchanger but does function as an electrogenic anion conductance, perhaps a channel. We have also cloned murine Slc26a9, which is strongly expressed in the respiratory tract and stomach. Radioisotope uptakes in Xenopus oocytes indicate that Slc26a9 is a highly selective anion exchanger, transporting Cl- but neither formate, oxalate, nor SO42-. We also utilized electrophysiology to voltage clamp (VC) and/or measure intracellular pH (pHi), Cl- ([Cl-],) and Na+ ([Na+]i), in response to various ion replacements. Cl- removal in HCO3- depolarizes oocytes (to > +60mV), alkalinizes oocytes, and decreases aCl-i. Slc26a9 thus functions as an electrogenic nCl-/HCO3- exchanger, suggesting a role in pulmonary and gastric HCO3- secretion and/or CO2 transport. VC experiments revealed channel-like currents (>10 microA at -60mV and >80 microA at +60mV) mediated by Slc26a9 in the presence and absence of HCO3-. Our experiments and those of others continue to reveal additional characteristics and unique roles for this new class of electrogenic anion transporters.

Renal urate transport.

Serum uric acid is determined by a balance between production and renal excretion. Luminal reabsorption of urate by the proximal tubule from the glomerular ultrafiltrate involves coupling between sodium-anion cotransport and urate-anion exchange. Apical sodium-coupled cotransport of lactate, ketoacids, nicotinate, and pyrazinoate increases intracellular levels of these anions in proximal tubular cells, stimulating the apical absorption of luminal urate via anion exchange. Hyperuricemia occurs when plasma levels of these anions increase; for example, hyperuricemia is a well-recognized concomitant of lactic acidosis and ketoacidosis. Relevant developments in the molecular and renal physiology of urate homeostasis are reviewed.

Primary and secondary prevention of chronic kidney disease.

The incidence and prevalence of chronic kidney disease (CKD) is on the rise worldwide. End-stage renal disease (ESRD) is the most advanced form of CKD, requiring some form of renal replacement therapy to ensure survival. Interventions to prevent or slow the progression of CKD, irrespective of the original cause, are thus of significant importance. The most effective of these interventions is based on the inhibition of the renin-angiotensin system (RAS) and is the main focus of this review.

Acetaminophen-induced anion gap metabolic acidosis and 5-oxoprolinuria (pyroglutamic aciduria) acquired in hospital.

A rare cause of high anion gap acidosis is 5-oxoproline (pyroglutamic acid), an organic acid intermediate of the gamma-glutamyl cycle. Acetaminophen and several other drugs have been implicated in the development of transient 5-oxoprolinemia in adults. We report the case of a patient with lymphoma who was admitted for salvage chemotherapy. The patient subsequently developed fever and neutropenia and was administered 20.8 g of acetaminophen during 10 days. During this time, anion gap increased from 14 to 30 mEq/L (14 to 30 mmol/L) and altered mental status developed. After usual causes of high anion gap acidosis were ruled out, a screen for urine organic acids showed 5-oxoproline levels elevated at 58-fold greater than normal values. Predisposing factors in this case included renal dysfunction and sepsis. Clinicians need to be aware of this unusual cause of anion gap acidosis because it may be more common than expected, early discontinuation of the offending agent is therapeutic, and administration of N -acetylcysteine could be beneficial.

Strategies to retard the progression of chronic kidney disease.

Chronic kidney disease (CKD) is a common and, in advanced cases, highly morbid disorder. The most severe form of CKD is end-stage renal disease (ESRD), in which the patient requires some form of renal replacement therapy to survive. The increasing incidence, prevalence, and costs of ESRD are major national health care concerns; interventions that may prevent or slow the progression of CKD towards ESRD are extremely important and the focus of this article.

Why is proteinuria an ominous biomarker of progressive kidney disease?

Progressive tubule injury and interstitial fibrosis frequently accompany glomerulopathies associated with proteinuria. Clinical experience indicates that higher levels of proteinuria prior to, as well as after initiation of treatment predict more rapid decline in renal function and more pronounced tubulointerstitial injury. It has been proposed that filtration of potentially tubulotoxic plasma proteins is responsible for the observed correlations between proteinuria and progression (i.e., proteinuria is a cause and not only a consequence of progressive renal injury). Numerous attempts have been made to identify the species of putative tubulotoxic proteins in this progressive injury process, but much uncertainty persists. These uncertainties stem from nonphysiologic exposure of apical cell surfaces to proteins in vitro, the extremely high concentrations of various proteins tested in vitro, and the nonuniformity of end points measured. Furthermore, there is often a lack of correlation between in vitro and in vivo findings, and a lack of uniformity of results even for seemingly similar in vitro experiments. Less controversy is evident in the potential pathways whereby injured tubules evoke a tubulointerstitial inflammatory and fibrotic response, with many in vivo models serving to incriminate excessive cytokine and chemokine production, infiltration of various inflammatory cells, and the balance between apoptosis and cell proliferation. Despite many years of concerted efforts, we believe it is still unclear whether proteinuria is a cause (and if so, which species of protein), or only a consequence of progressive renal injury. Nevertheless, pending the resolution of these uncertainties by more decisive and unambiguous experimentation, the strongly predictive inverse relationship between level of proteinuria and long-term renal survival currently justifies aggressive antiproteinuric treatment strategies, with a goal of reducing protein excretion rate to the lowest level possible without the induction of symptoms or undue risk.

The independent association between serum uric acid and graft outcomes after kidney transplantation.

BACKGROUND: Improving long-term outcomes of kidney transplantation depends on identifying novel risk factors that lead to poor outcomes. We sought to evaluate the predictive value of mean uric acid (UA) level during the first 6 months posttransplant for graft survival and function. METHODS: Two hundred twelve recipients of living donor kidneys transplanted during January 2000 to December 2001 were included. The study outcome included graft and patient survival and graft function at 1 year posttransplant. Regression models were used to adjust for the confounding variables including graft function during first 6 months. RESULTS: During 68.3 + or - 27.2 months follow-up, UA level (mg/dL) and hyperuricemia (n=45) were associated with graft loss (hazard ratio [HR]=1.26, P=0.026, 95% confidence interval [CI]=1.03-1.53, and HR=1.92, P=0.029, 95% CI=1.1-3.4, respectively) independent of graft function and other confounders. UA also seemed to be associated with risk of death with borderline significance (HR=1.2, P=0.096, 95% CI=0.97-1.46). Examining the predictive value for graft function, UA level and hyperuricemia were independent predictors of 1-year serum creatinine (beta=0.10, P=0.013, 95% CI=0.02-0.18, and beta=0.25, P<0.04, 95% CI=0.01-0.49, respectively). Similarly, both were associated with 1-year estimated glomerular filtration rate (beta=-3.9, P<0.001, 95% CI=-5.7 to -1.5 for UA, and beta=-7.6, P<0.02, 95% CI=-13.6 to -1.5 for hyperuricemia). Notably, these associations were all independent of renal function during first 6 months. CONCLUSION: The results of this study suggest that mean UA level during the first 6 months posttransplant is an independent predictor of long-term graft survival and short-term graft function. Further investigations are needed to evaluate its causal association with chronic allograft injury and cardiovascular disease.

Adult hypertension and kidney disease: the role of fetal programming.

Hypertension (HTN) and chronic kidney disease are highly prevalent diseases that tend to occur more frequently among disadvantaged populations, in whom prenatal care also tends to be poor. More and more evidence is emerging highlighting the important role of fetal programming in the development of adult disease, suggesting a possible common pathophysiologic denominator in the development of these disorders. Epidemiologic evidence accumulated over the past 2 decades has demonstrated an association between low birth weight and subsequent adult HTN, diabetes, and cardiovascular disease. More recently, a similar association has been found with chronic kidney disease. Animal studies and indirect evidence from human studies support the hypothesis that low birth weight, as a marker of adverse intrauterine circumstances, is associated with a congenital deficit in nephron number. The precise mechanism of the reduction in nephron number has not been established, but several hypotheses have been put forward, including changes in DNA methylation, increased apoptosis in the developing kidney, alterations in renal renin-angiotensin system activity, and increased fetal glucocorticoid exposure. A reduction in nephron number is associated with compensatory glomerular hypertrophy and an increased susceptibility to renal disease progression. HTN in low birth weight individuals also appears to be mediated in part through a reduction in nephron number. Increased awareness of the implications of low birth weight and inadequate prenatal care should lead to public health policies that may have long-term benefits in curbing the epidemics of HTN, diabetes, and kidney disease in generations to come.